Title

Author

Date of Award

2006

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Walter A. Scott, Committee Chair

Abstract

The formation of RT complex induced by the next complementary dNTP was observed and is referred to as the +1 complex. It was identified as a stable by various biochemical assays and forms cross-links at a specific position in the template upon UV treatment.A unique RT complex formed in the presence of PFA (phosphonoformic acid or foscarnet, a pyrophosphate analog) was also observed 1 nt upstream compared to the +1 complex, indicating that the PFA complex stays in a pre translocated position whereas the +1 complex stays in a post-translocated position.Another unique, stable complex formed with the dNTP complementary to the +2 base of the template, named the +2 complex, was detected. The +2 complex as predominantly positioned 1 nt downstream from the +1 complex which places RT in a "hyper-translocated" position. The +2 complex is less stable than the +1 and PFA complexes. Differences in positioning of the +1, +2 and PFA complexes on DNA indicate that binding of the +1 dNTP, the +2 dNTP or PFA can control positioning of RT on DNA.One of the distinctive characteristics of the +1, +2 and PFA complexes is the tight interaction of the downstream border of RT on the template DNA. Furthermore, the specific interaction between RT and the single-stranded portion of the template was identified as the +2 base of the template in the +1 complex, which is consistent with the crystal structure of Huang et al. (1998). More importantly, the interaction of RT with the +1 base of the template observed in the PFA cross-linked product reinforced the conclusion that the PFA complex captures RT in a "closed" conformation and is positioned 1 nt upstream on DNA compared to the +1 complex.The characterization of stable complex formation by HIV-1 RT induced by the binding of dNTPs or PFA yielded new insights into the mechanistic properties of HIV-1 RT that are most likely involved in conformational change, translocation, fidelity, processivity and possible product inhibition by PFA.